本试剂盒适合于从动物组织、细胞、植物、细菌和真菌等样本中快速分离RNA，为动物组织和细胞等生物样本总RNA提取提供了一个简单快速的解决方案。试剂盒基于超顺磁性的磁性粒子纯化技术，无需进行耗时的醇类沉淀。得到的RNA可直接用于RT-PCR、Northern Blot、Poly A纯化、核酸保护和体外翻译等实验。其处理后的产物用于科研检测使用。
储存条件
M1521678A, M1521678B：2-8℃避光保存，12个月。
M1521678C：2-8℃保存，12个月。
M1521678D, M1521678E, M1521678F：室温保存，12个月。
适配仪器
手工提取
注意事项
1. 使用前请仔细阅读本说明书。
2. 本试剂盒中的多种缓冲液含有刺激性的胍盐，务必戴上手套，并按照安全标准预防措施来处理。不要让缓冲液接触到皮肤、眼睛以及黏膜，如果确实发生，请立即用大量清水清洗并就医。
3. 如因气温较低，溶液出现沉淀，需要30℃水浴至沉淀完全溶解后方可使用。
4. 如因气温较低，用力振荡后磁珠无法重悬，请勿使用。
5. 本试剂盒中的多种缓冲液含胍盐，请勿用氧化性消毒剂如次氯酸钠进行处理，否则会释放有毒气体，须按医疗废物进行处理。
6. 本试剂盒需配套使用，不同批号试剂盒中各组分不可以互换或混用。
使用方法
1. 样本要求
- 适用标本类型：动物组织、细胞、植物、细菌和真菌等样本。
- 标本保存和运输：标本可立即用于测试，也可以保存于-70℃或更低温度待测，保存期为6个月，应避免反复冻融。标本运输采用冷链运输。
- 冻融要求：速冻速融，避免反复冻融。
2. 自备试验器材与试剂
- 恒温水浴锅（当溶液出现沉淀时使用）、组织匀浆器、2 mL匀浆管
- 磁力架（1.5/2.0 mL）、1.5/2.0 mL 无菌无核酸酶离心管、各种规格移液器及Tips（20、200、1000 μL）
- 无水乙醇
3. 试剂准备
4. 操作步骤
磁分离操作建议（步骤4.6开始）：离心管置于磁力架后，轻轻地左右旋转，待磁珠聚集于贴近磁力架的管壁后，轻轻地颠倒磁力架数次，使管盖上的磁珠也聚集到管壁，静置数分钟至溶液澄清即可（静置时长视磁力架磁性而定）。
4.1 根据不同样本类型选择对应步骤进行处理
(1) 组织
- 称取约20 mg动物组织样本于1.5 mL离心管，加入1 mL 总RNA提取试剂后用组织匀浆器进行匀浆，室温静置5分钟，使其充分裂解。
- 取适量动物组织样本，使用液氮充分研磨后，称取约20 mg于1.5 mL离心管，加入1 mL 总RNA提取试剂，涡旋打散样本，室温静置5分钟，使其充分裂解。
(2) 细胞
- 培养贴壁细胞：≤5× 10⁶个培养细胞，彻底吸弃培养基后，加入1 mL 总RNA提取试剂，使用移液器吸打3~5次进行消化，全部转移至1.5 mL离心管中，室温静置5分钟，使其充分裂解。
- 悬浮细胞：500×g离心5分钟收集细胞（≤5× 10⁶个细胞），彻底去除培养基，涡旋或用手指弹打使细胞分散，加入1 mL 总RNA提取试剂，使用移液器吸打3~5次进行消化、裂解，室温静置5分钟，使其充分裂解。
(3) 植物
用液氮将植物样本研磨成粉末状，称取50~100 mg的样本至1.5 mL离心管中，立即加入1 mL 总RNA提取试剂，涡旋打散样本。室温静置5分钟，使其充分裂解。
(4) 细菌
离心收集（1× 10⁸细菌），加入100 µL TE/lysozyme（自备）处理10分钟，然后加入1 mL 总RNA提取试剂，涡旋1分钟。
(5) 微量真菌
转移10~20 mg真菌样本至2 mL匀浆管中，加入1 mL 总RNA提取试剂，高速涡旋5~10分钟，使其充分裂解。
4.2 （可选）4℃，12,000×g离心10分钟后取全部上清于新的离心管中。
备注：处理富含蛋白质或难裂解的组织样本时，匀浆后可能仍有未溶解的物质。离心去除这些杂质有利于提高纯度。富含脂肪的样本，离心后还会在溶液表面存在一层油脂层，转移上清液时尽量不要吸取到油脂。
4.3 加入200 µL的分层液SF或氯仿，用手剧烈振荡15秒（请勿使用涡旋仪涡旋），室温放置3~5分钟。
备注：用涡旋取代振荡会带来更多的基因组DNA污染。振荡必须快速的，缓慢颠倒会造成抽提不充分，分层液SF为氯仿替代试剂，须按比例加入，过多的分层液SF会使DNA和蛋白质回到水相中，导致RNA的纯度下降。若有自备氯仿，也可使用氯仿进行操作。
4.4 4℃，12,000×g离心15分钟。
备注：离心后会形成三相体系。上层为水相层含有RNA，而DNA和蛋白质位于中间层和有机层（下层）。中间层的多少取决于样本类型，有些样本的中间层会较少。
4.5 小心吸取上清水相至新的离心管中，加入等体积无水乙醇和20 μL磁珠MXVB，振荡或涡旋10分钟使磁珠吸附RNA。
4.6 转移至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.7 加入700 µL洗涤液DW1，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.8 加入700 µL洗涤液DW2，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.9 再次加入700 µL洗涤液DW2，涡旋30秒后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.10 掌上离心机瞬时离心后，转至磁力架上吸附至溶液澄清，充分吸弃所有溶液，打开管盖，空气干燥3~5分钟。
注意：乙醇残留会抑制后续的酶反应，所以晾干时要确保乙醇挥发干净。切勿干燥过久，以免影响后续洗脱效果。
4.11 加入50~100 µL洗脱液RE，高速涡旋2~3分钟打散磁珠。
4.12 瞬时离心收集管盖液滴至管中，转移至磁力架上进行磁分离。
4.13 提取的RNA请转移至1.5 mL无菌无核酸酶的离心管中，如果不立即使用，请存储于-15~-25℃，长期保存请放置于-70℃或更低的温度。
方法的局限性
1. 对于某些取样不佳的样本，提取产物中可能存在少量杂质抑制PCR扩增。
2. 样本核酸的提取效果与样本收集、处理、运输和保存质量密切相关，其中任何失误都将会导致结果不准确。
This kit is designed for the rapid isolation of RNA from various sample types, including animal tissues, cells, plants, bacteria, and fungi. It provides a simple and rapid solution for total RNA extraction from biological samples such as animal tissues and cells. The kit is based on superparamagnetic particle purification technology, eliminating the need for time-consuming alcohol precipitation. The resulting RNA can be used directly in downstream applications such as RT-PCR, Northern Blot, Poly A purification, nuclease protection assays, and in vitro translation. The processed products are intended for scientific research and testing purposes only.
Storage Conditions
M1521678A, M1521678B: Store at 2–8°C protected from light, stable for 12 months.
M1521678C: Store at 2–8°C, stable for 12 months.
M1521678D, M1521678E, M1521678F: Store at room temperature, stable for 12 months.
Compatible Instruments
Manual extraction.
Precautions
Read this manual carefully before use.
Several buffers in this kit contain irritating guanidinium salts. Always wear gloves and handle according to standard safety precautions. Avoid contact of buffers with skin, eyes, and mucous membranes. In case of contact, flush immediately with plenty of water and seek medical attention.
If precipitation occurs in the solution due to low ambient temperatures, incubate at 30°C in a water bath until the precipitate is completely dissolved before use.
If the magnetic beads cannot be resuspended after vigorous shaking due to low ambient temperatures, do not use the beads.
Several buffers in this kit contain guanidinium salts. Do not treat with oxidizing disinfectants such as sodium hypochlorite, as this may release toxic gases. Dispose of waste according to medical waste regulations.
This kit components are designed to be used together as a system. Components from different lot numbers should not be interchanged or mixed.
Instructions for Use
1. Sample Requirements
Applicable sample types: Animal tissues, cells, plants, bacteria, fungi, and similar samples.
Sample storage and transport: Samples may be processed immediately or stored at -70°C or lower for up to 6 months. Avoid repeated freeze-thaw cycles. Transport samples using cold chain logistics.
Freeze-thaw requirements: Rapid freezing and rapid thawing are recommended; avoid repeated freeze-thaw cycles.
2. Equipment and Reagents to be Supplied by User
Constant temperature water bath (for use when precipitation occurs in solutions)
Tissue homogenizer
2 mL homogenization tubes
Magnetic separation rack (for 1.5/2.0 mL tubes)
1.5/2.0 mL sterile, nuclease-free centrifuge tubes
Pipettes and tips of various volumes (20, 200, 1000 μL)
Absolute ethanol
3. Reagent Preparation
Check solutions for any precipitation and confirm that the magnetic beads can be resuspended.
4. Operating Procedure
Recommendation for magnetic separation (starting from step 4.6): After placing the centrifuge tube on the magnetic rack, gently rotate it left and right. Once the magnetic beads have aggregated against the tube wall closest to the magnet, gently invert the entire rack several times to collect any beads from the cap onto the tube wall. Allow to stand for several minutes until the solution becomes clear (standing time depends on the strength of the magnetic rack).
4.1 Select the appropriate procedure based on sample type:
(1) Tissue
Weigh approximately 20 mg of animal tissue sample into a 1.5 mL centrifuge tube. Add 1 mL of Total RNA Extraction Reagent and homogenize using a tissue homogenizer. Let stand at room temperature for 5 minutes to ensure complete lysis.
Alternatively, grind an appropriate amount of animal tissue sample thoroughly using liquid nitrogen. Weigh approximately 20 mg of the ground powder into a 1.5 mL centrifuge tube. Add 1 mL of Total RNA Extraction Reagent, vortex to disperse the sample, and let stand at room temperature for 5 minutes to ensure complete lysis.
(2) Cells
Adherent cultured cells: For ≤ 5 × 10⁶ cultured cells, completely aspirate the culture medium. Add 1 mL of Total RNA Extraction Reagent and pipette up and down 3–5 times to digest the cells. Transfer the entire lysate to a 1.5 mL centrifuge tube and let stand at room temperature for 5 minutes to ensure complete lysis.
Suspension cells: Centrifuge at 500 × g for 5 minutes to collect the cells (≤ 5 × 10⁶ cells). Completely remove the culture medium. Vortex or flick the tube to disperse the cell pellet. Add 1 mL of Total RNA Extraction Reagent and pipette up and down 3–5 times for digestion and lysis. Let stand at room temperature for 5 minutes to ensure complete lysis.
(3) Plant
Grind the plant sample into a fine powder using liquid nitrogen. Weigh 50–100 mg of the sample powder into a 1.5 mL centrifuge tube. Immediately add 1 mL of Total RNA Extraction Reagent and vortex to disperse the sample. Let stand at room temperature for 5 minutes to ensure complete lysis.
(4) Bacteria
Collect bacteria by centrifugation (1 × 10⁸ cells). Add 100 μL of TE buffer containing lysozyme (user-supplied) and incubate for 10 minutes. Then, add 1 mL of Total RNA Extraction Reagent and vortex for 1 minute.
(5) Micro-fungi
Transfer 10–20 mg of fungal sample to a 2 mL homogenization tube. Add 1 mL of Total RNA Extraction Reagent and vortex at high speed for 5–10 minutes to ensure complete lysis.
4.2 (Optional) Centrifuge at 12,000 × g for 10 minutes at 4°C, then transfer all of the supernatant to a new centrifuge tube.
Note: When processing samples rich in protein or difficult-to-lyse tissues, undissolved material may remain after homogenization. Centrifugation to remove this debris helps improve purity. For lipid-rich samples, a layer of lipid may appear on the surface of the solution after centrifugation; avoid aspirating this lipid layer when transferring the supernatant.
4.3 Add 200 μL of Phase Separation Buffer SF or chloroform. Shake vigorously by hand for 15 seconds (do not use a vortex mixer). Let stand at room temperature for 3–5 minutes.
Note: Using a vortex mixer instead of manual shaking may result in increased genomic DNA contamination. Shaking must be vigorous; gentle inversion will lead to insufficient phase separation. Phase Separation Buffer SF is a chloroform substitute and must be added in the specified ratio. Excessive amounts of SF may cause DNA and protein to re-enter the aqueous phase, reducing RNA purity. If chloroform is available, it may be used for this step instead.
4.4 Centrifuge at 12,000 × g for 15 minutes at 4°C.
Note: After centrifugation, a three-phase system will form. The upper aqueous phase contains RNA, while DNA and proteins are located in the interphase and the lower organic phase. The thickness of the interphase varies depending on the sample type and may be minimal for some samples.
4.5 Carefully aspirate the upper aqueous phase and transfer it to a new centrifuge tube. Add an equal volume of absolute ethanol and 20 μL of Magnetic Beads MXVB. Shake or vortex for 10 minutes to allow RNA binding to the beads.
4.6 Place the tube on the magnetic rack for magnetic separation until the solution is clear, then aspirate and discard the supernatant.
4.7 Add 700 μL of Wash Buffer DW1. Vortex for 30 seconds, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.8 Add 700 μL of Wash Buffer DW2. Vortex for 30 seconds, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.9 Add another 700 μL of Wash Buffer DW2. Vortex for 30 seconds, then place on the magnetic rack for separation until the solution is clear. Aspirate and discard the supernatant.
4.10 Perform a quick spin using a mini centrifuge, then place the tube back on the magnetic rack and allow beads to collect until the solution is clear. Carefully aspirate and discard all residual liquid. Open the tube cap and air-dry the beads for 3–5 minutes.
Note: Residual ethanol will inhibit downstream enzymatic reactions. Ensure ethanol has completely evaporated during drying. Do not over-dry the beads, as this may affect subsequent elution efficiency.
4.11 Add 50–100 μL of Elution Buffer RE and vortex at high speed for 2–3 minutes to fully disperse the beads.
4.12 Briefly centrifuge to collect any droplets from the cap into the tube, then place on the magnetic rack for magnetic separation.
4.13 Transfer the extracted RNA solution to a new 1.5 mL sterile, nuclease-free centrifuge tube. If not used immediately, store at -15 to -25°C. For long-term storage, place at -70°C or lower.
Limitations of the Method
For certain poorly collected samples, trace impurities in the extracted product may inhibit PCR amplification.
The efficiency of nucleic acid extraction is closely related to the quality of sample collection, handling, transport, and storage. Any error in these processes may lead to inaccurate results.
ZJ26F0636400
危险品化学品经营许可证（带存储）